Oral formulations of mitochondrially-targeted antioxidants and their preparation and use

ABSTRACT

Provided are stable liquid and solid formulations of oxidized and reduced mitochondria-targeted antioxidants, and methods of their preparation and use.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the national phase under 35 U.S.C. §371 ofInternational Application No. PCT/US2012/040711, entitled “OralFormulations of Mitochondrially-Targeted Antioxidants and TheirPreparation and Use,” filed on Jun. 4, 2012, which claims priority toand the benefit of U.S. Provisional Patent application Ser. No.61/492,940 entitled “Oral Formulations of Mitochondrially-TargetedAntioxidants and Their Medical Use” which was filed Jun. 3, 2011. Theentirety of the aforementioned applications are herein incorporated byreference.

FIELD OF THE INVENTION

This disclosure is in the fields of cell biology, pharmacology andmedicine, and in particular, inflammation, diabetes, septic shock, woundhealing, and coronary heart disease.

BACKGROUND

Promising therapeutical properties of mitochondria-targeted antioxidants(MTAs) have been described (see, e.g., US2008176929; Skulachev et al.(2009), Biochim. Biophys. Acta, 1787:437-61). The experiments performedwhich revealed these properties were done with freshly preparedsolutions of MTAs and made by dissolving of ethanol stock solutionspreserved at −80° C. shortly before administration of the preparation toanimals. Such method of preparation and administration is not suitableor realistic for preparation of pharmaceuticals as it is extremelyinconvenient if not impossible for industrial manufacturing, logistics,and use by patients. Attempts to develop a pharmaceutical composition(for oral administration or injection) with acceptable stabilityrevealed that MTAs are not stable in most types of oral or injectablecompositions. Stable pharmaceutical composition containing MTAspossessing acceptable stability have not been described up to now.Accordingly, improved liquid formulations with stability are stillneeded.

SUMMARY

The present disclosure provides stabilized liquid and solid formulationscomprising MTAs suitable for oral, nasal, and intravenous and injectableadministration, and methods of preparation of such formulations. Theinvention also provides methods of treatment and prophylaxis of diseasesand conditions relating to mitochondria using such formulations.

In one aspect, the disclosure provides a stabilized pharmaceuticalformulation comprising a compound of Formula I in oxidized and/orreduced form.

The compound of Formula I is:

wherein:

A is an antioxidant of Formula II:

and/or reduced form thereof, wherein m comprises an integer from 1 to 3;

Y is independently selected from the group consisting of: lower alkyl,lower alkoxy, or two adjacent Y groups, together with carbon atoms towhich they are attached, form a following structure of Formula III:

and/or reduced form thereof, wherein:

R1 and R2 are the same or different and are each independently loweralkyl or lower alkoxy;

L is a linker group, comprising: a) a straight or branched hydrocarbonchain optionally substituted by one or more double or triple bond, orether bond, or ester bond, or C—S, or S—S, or peptide bond; and which isoptionally substituted by one or more substituents preferably selectedfrom alkyl, alkoxy, halogen, keto group, amino group; or b) a naturalisoprene chain;

n is an integer from 1 to 20; and

B is a targeting group comprising: a) a Skulachev-ion Sk (Sk⁺Z⁻)wherein: Sk is a lipophillic cation or a lipophillic metalloporphyrin,and Z is a pharmaceutically acceptable anion; or b) an amphiphilliczwitterion,

with the proviso that in compound of Formula I, A is not ubiquinone(e.g., 2-methyl-4,5-dimethoxy-3,6-dioxo-1,4-cyclohexadienyl) ortocopherol or a mimetic of superoxide dismutase or ebselen; when L isdivalent decyl, divalent pentyl, or divalent propyl radical; and when Bis triphenylphosphonium cation.

In a particular embodiment, the composition is reduced or is oxidized.In some embodiments, the formulation is in liquid form, and in otherembodiments, the formulation is in solid form.

In some embodiments the liquid formulation comprises a compound ofFormula I in 10% to 100% glycerol, from about 10% to about 100% glycol,(e.g., 1,2-propylene glycol) or from about 1% to about 100% (absolute)ethanol. In one particular embodiment, the composition of Formula I isin about 50% 1,2-propylene glycol.

The disclosure also provides stabilized solid pharmaceuticalformulations comprising a compound of Formula I in oxidized or reducedform, with the proviso that in compound of Formula I, A is notubiquinone (e.g., 2-methyl-4,5-dimethoxy-3,6-dioxo-1,4-cyclohexadienyl)or tocopherol or a mimetic of superoxide dismutase or ebselen; when L isdivalent decyl, divalent pentyl, or divalent propyl radical; and when Bis triphenylphosphonium cation.

In one embodiment, the formulation also comprises 1 molar equivalent to200 molar equivalents of an antioxidation agent that reduces theoxidized form of the compound of Formula 1, and a pharmaceuticallyacceptable carrier.

In some embodiments, the antioxidation agent is ascorbic acid.

In some embodiments, the pharmaceutically acceptable carrier comprisessorbite, glucose, and/or magnesium stearate.

In certain embodiments, the pharmaceutical formulation is SkQ1 orSkQ1H₂. In other embodiments, the compound is SkQR1 or SkQR1H₂. In yetother embodiments, the compound is SkQ3 or SkQ3H₂. In still otherembodiments, the compound is SkQRB or SkQRBH₂. In other embodiments, thecompound is SkQB1 or SkQB1H₂. In yet other embodiments, the compound isSkQBP1 or SkQBP1H₂.

In other aspects, the disclosure provides methods of treating andpreventing diabetes type I and II, inflammation, septic shock,arthritis, and coronary heart disease, and methods of aiding in woundhealing. In these methods, a therapeutically effective amount of aformulation comprising a stabilized compound of Formula I in liquid orsolid form is administered to a patient, with the proviso that incompound of Formula I, A is not ubiquinone (e.g.,2-methyl-4,5-dimethoxy-3,6-dioxo-1,4-cyclohexadienyl) or tocopherol or amimetic of superoxide dismutase or ebselen; when L is divalent decyl,divalent pentyl, or divalent propyl radical, and when B istriphenylphosphonium cation.

In some embodiments of the method, the formulation comprises glycerol,glycol, and/or ethanol. In some embodiments, the formulation comprisesSkQ1, SkQ1H₂, SkQR1, SkQR1H₂, SkQ3, SkQ3H₂, SkQBP1, SkQBP1H₂, SkQRB, orSkQRBH₂.

In some embodiments, the liquid formulation is administered orally or byinjection. In other embodiments, the solid formulation is administeredorally, anally, or vaginally. In some embodiments the formulation is asolid and comprises ascorbic acid. In particular embodiments, theformulation also comprises a pharmaceutically acceptable carrier.

In some embodiments, diabetes type I or II is treated with SkQ1 orSkQ1H₂ in 20% glycerol.

In certain embodiments, arthritis is treated with a formulationcomprising SkQ1 or SkQ1H₂ in 20% glycerol. In yet other embodiments,arthritis is treated with a formulation comprising SkQ1 and ascorbicacid.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of the present disclosure, the variousfeatures thereof, as well as the invention itself may be more fullyunderstood from the following description, when read together with theaccompanying drawings.

FIG. 1 is a graphic representation of the effect of SkQ1 on bloodglucose level of diabetic animal model (alloxan-treated mice);

FIG. 2 is a graphic representation of the effect of SkQ1 on liver damageof db/db diabetic mice;

FIG. 3 a is a graphic representation illustrating the effect of SkQ1 onepithelization of diabetic wounds;

FIG. 3 b is a graphic representation illustrating the effect of SkQ1 onthe amount of neutrophils in diabetic wounds;

FIG. 3 c is a graphic representation illustrating the effect of SkQ1 onvessel density in diabetic wounds;

FIG. 4 is a graphic representation of the effect of SkQ1 on survival ofmice subjected to septic shock;

FIG. 5 is a graphic representation demonstrating the anti-inflammatoryeffect of SkQ1 in collagen-induced arthritis in rats;

FIG. 6 is a graphic representation demonstrating the anti-inflammatoryeffect of SkQ1 and SkQR1 rescuing endothelial cells from death inducedby proinflammatory cytokine TNF-alpha;

FIG. 7 a is a graphic representation demonstrating the ability of SkQ1to inhibit inflammation in vitro by lowering expression ofpro-inflammatory cytokines; and

FIG. 7 b is a graphic representation demonstrating the ability of SkQ1to inhibit inflammation in vivo by lowering expression ofpro-inflammatory cytokines as measured by relative ICAM-1 mRNAexpression in mice.

DESCRIPTION

Throughout the text of a description of the invention various documentsare cited. Each document cited here (including all patents, patentapplications, scientific publications, specifications and manufacturer'sinstructions etc.), above or below, is introduced in full in thisinvention by reference.

Prior to the detailed description of the invention follows, one shouldunderstand that the invention is not limited to the particularmethodology, protocols, and reagents described here, as they are subjectto change. In addition, it should be understood that in the presentinvention, the terminology is used to describe particular embodimentsonly and does not limit the scope of the present invention which will belimited only by the appended claims. Unless otherwise specified, alltechnical and scientific terms used here have the same meanings that areunderstandable to those skilled in the art.

It was unexpectedly found that many effective MTAs are not stable enoughin usual liquid and solid pharmaceutical formulations suitable for theiradministration by injection, or by oral, IV, nasal, topical, or enteraladministration. This feature limits clinical application ofpharmaceuticals based on MTA as active compounds.

I. Stabilized Formulations

The present disclosure provides stable, liquid, MTA-based pharmaceuticalcompositions applicable in clinical practice. A useful MTA is a compoundof Formula I in oxidized and/or reduced form.

The compound of Formula I is:

wherein:

A is an antioxidant of Formula II:

and/or reduced form thereof, wherein m comprises an integer from 1 to 3;

Y is independently selected from the group consisting of: lower alkyl,lower alkoxy, or two adjacent Y groups, together with carbon atoms towhich they are attached, form a following structure of Formula III:

and/or reduced form thereof, wherein:

R1 and R2 are the same or different and are each independently loweralkyl or lower alkoxy;

L is a linker group, comprising: a) a straight or branched hydrocarbonchain optionally substituted by one or more double or triple bond, orether bond, or ester bond, or C—S, or S—S, or peptide bond; and which isoptionally substituted by one or more substituents preferably selectedfrom alkyl, alkoxy, halogen, keto group, amino group; or b) a naturalisoprene chain;

n is an integer from 1 to 20; and

B is a targeting group comprising: a) a Skulachev-ion Sk: (Sk⁺Z⁻),wherein: Sk is a lipophillic cation or a lipophillic metalloporphyrin,and Z is a pharmaceutically acceptable anion; or b) an amphiphilliczwitterion, with the proviso that in compound of Formula I, A is notubiquinone (e.g., 2-methyl-4,5-dimethoxy-3,6-dioxo-1,4-cyclohexadienyl)or tocopherol or a mimetic of superoxide dismutase or ebselen; when L isdivalent decyl, divalent pentyl, or divalent propyl radical; and when Bis triphenylphosphonium cation, with the proviso that in compound ofFormula I, A is not ubiquinone (e.g.,2-methyl-4,5-dimethoxy-3,6-dioxo-1,4-cyclohexadienyl) or tocopherol or amimetic of superoxide dismutase or ebselen; when L is divalent decyl,divalent pentyl, or divalent propyl radical; and when B istriphenylphosphonium cation.

Specific useful MTAs include, but are not limited to, the SkQ1 andSkQR1:

and their reduced (quinole) forms SkQ1H₂ and SkQR1H₂, respectively.These MTAs have been described in PCT/RU2006/000394.

Other useful MTA variants include, but are not limited to SkQ3:

and its reduced (quinole) form SkQ3H₂;

to SkQRB:

and its oxydized (quinone) form SkQRB;

to SkQB1:

and its reduced (quinole) form, SkQB1H₂; and

to SkQBP1:

and its reduced (quinole) form SkQBP1H₂.

These MTAs are formulated for oral administration as liquid solutionsand as solid formulations.

Liquid solutions are also useful for aerosol delivery via injection, forIV administration, nasal administration, topical administration, orenteral administration.

Such stable liquid formulations include one or more solvents or solublecomponents into which the MTAs are placed. Useful solvents includeglycerol, ethanol, propyleneglycol, and analogous compounds. Forexample, useful stable formulations contain at least 10% 1,2-propyleneglycol, at least 1% or at least 10% ethanol, at least 10% glycerol, ormixtures thereof, which may also include water, glycerol, ethanol,and/or 1,2-propylene to make up the difference. For example,representative stabilizing solutions of 1 nM to 1 mM SkQ1, SkQ1H₂,SkQR1, SkQR1H₂, SKQ3, SkQ3H₂, SKQRB, SkQRBH₂, SKQB1, SkQB1H₂, SKQBP1and/or SkQBP1H₂, contain 10% to 50%, 50% to 100%, 10% to 20%, 20% to30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to90%, 10% to 100%, 20% to 80%, and 90% to 100% 1,2-propylene glycol,glycerol, or ethanol. Other useful percentages of such solvents include15%, 20%, 25%, 30%, 35%, 40%, 45%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, and 95%. Other pharmaceutically acceptable carriers may also becomponents of such formulations.

Because MTAs are not shelf-stable for long periods of time, variouscompounds were tested to determine their ability to stabilize SkQ1 andSkQR1 as representative MTAs in dry form.

Beta-cyclodextrin, gun-arabic, fruit fibers, and sodium chloride did notprovide suitable stabilization levels (degradation rate, %/d was 0.8 to8.1).

Liquid solvents were also tested for their ability to stabilizerepresentative MTAs SkQ1 and SkQR1. The solvents tested were watersolutions of glycerol (10% to 100%), 50% lactulose, and 1,2-propyleneglycol (10% to 100%, at 60° C.). Some representative results are shownbelow (Table 1).

TABLE 1 Degradation Concentration, Stabilizing rate, percent MTA mkMSolvent per day SkQ1 400  50% lactulose 9.01 SkQ1 400  10% 1,2-propyleneglycol 0.47 SkQ1 400  50% 1,2-propylene glycol 0.06 SkQ1 400 100%1,2-propylene glycol 0.18 SkQ1 400  10% Glycerol 0.61 SkQ1 400  20%Glycerol 0.51 SkQ1 400  30% Glycerol 0.53 SkQ1 400  40% Glycerol 0.91SkQ1 400  50% Glycerol 1.54 SkQ1 400  60% Glycerol 1.92 SkQ1 400  70%Glycerol 2.4 SkQ1 400  80% Glycerol 3.2 SkQ1 400  90% Glycerol 4.18SkQRB 200  50% Glycerol 0.4 SkQR1 140  50% Glycerol 0.7 SkQBP1 400  50%Glycerol 0.08 SkQR1 100  10% 1,2-propylene glycol 6.19 SkQR1 100  20%1,2-propylene glycol 0.34 SkQR1 100  30% 1,2-propylene glycol 0.32 SkQR1100  40% 1,2-propylene glycol 0.06 SkQR1 100  50% 1,2-propylene glycol<0.01 SkQR1 100  60% 1,2-propylene glycol <0.01 SkQR1 100  70%1,2-propylene glycol 0.05 SkQR1 100  80% 1,2-propylene glycol 0.23 SkQR1100  90% 1,2-propylene glycol 0.30 SkQR1 100 100% 1,2-propylene glycol0.23

These results illustrate high stability of MTAs in a pharmaceuticalcomposition for administration in the form of solution in glycerol (fromabout 10% to about 100% glycerol), and about 50% 1,2-propylene glycolsolution.

In addition, the stability of SkQ1 and SkQR1 was significantly increasedin dark plastic or glass vials, indicating that these compounds arelight-sensitive. Accordingly, one of the ways to further improve orincrease stability of SkQ liquid compositions during storage andtransportation is to protect it from light.

When SkQ compounds of Formula I according to the disclosure are in solidform, they may be stabilized, for example, with an antioxidation agent.Such an agent can be ascorbic acid. Useful amounts of ascorbic acidrange from about 1 molar equivalent to about 200 molar equivalents. Asused herein, the term “molar equivalent” refers to the number ofdissolved particles, or that amount which reacts with, or supplies onemole of H⁺ in an acid-base reaction, or which reacts or supplies onemole of electrons in a redox reaction. Other useful components ofrepresentative stabilized MTA formulations are shown in Table 2. Suchformulations may also comprise pharmaceutically acceptable carriers suchas, but not limited to, sorbite, glucose, and magnesium stearate.

Another approach to stabilize an SkQ compound in a pharmaceuticalformulation is to use its reduced (quinole) form. For example, thereduced form of SkQ1 is the quinole SkQ1H₂:

where Z⁻ is pharmaceutically acceptable anion such as, but not limitedto, bromide, chloride, or ascorbate. In a dry or soluble pharmaceuticalcomposition SkQ1H₂ can be stabilized and protected from oxidation by areducing agent such as, but not limited to, ascorbate.

Yet another approach to improve stability is to place the MTA, inreduced or oxidized form, in a “softgel” formulation, which is agelatin-based capsule with a liquid filling. Softgel formulations ofMTAs provide good bioavailability as the softgel dissolves inaqueous-miscible, oily liquid carriers such as mono- and digycerides ofcapric/caprylic acid (Capmul MCM), Miglyol oil 8122 (medium chaintriglycerides). When the softgel is released in the body, it getsemulsified and provides drug dispersion at a high surface area.

Mono- and digycerides of capric/caprylic acid (Capmul MCM), Miglyol oil8122 (medium chain triglycerides) can be used. Such oily carriers asthey become part of a self-emulsifying system. Other exemplarystabilizing components are vitamin E/polyethylene glycol succinate,sorbitan monooleate, labrasol, and combinations thereof. Additionally,based on its oxidation potential, tocopherol, butylayed hydroxytoluene,and/or butylated hydroxy anisole can be included in the composition asan antioxidant.

Another approach for increasing stabilization of MTAs in solution is tocreate a nanosuspension of MTA (<1000 nm) stabilized with, e.g., vitaminE/polyethylene glycol succinate. Netzsch wet milling(http://www.netzsch-grinding.com) can be used to achieve thisnanosuspension.

Additionally, ethanol solutions of reduced MTA (such as SkQ1H₂) can bemixed with the asorbic and acid dried to create resulting solid orpowder that is stable for several months.

Stable formulations in the form of oral tablets can be prepared by hotmelt extrusion. This melt granulation technique maintains thepolymorphic stability of the drugs and significantly improve their oralbioavailability. It can be achieved by co-blending the MTAs withmacrogols (e.g., polyethylene glycols 3350, 6000, polyvinyl pyrrolidone,hydroxy propyl cellulose and Vitamin E TPSG) through a hot meltextruder, and compressing the resulting granulation into tablets orencapsulating into hard gelatin capsules.

Representative stable liquid and solid oral SkQ1 formulations are shownbelow (Table 2):

TABLE 2 Oxidized SkQ1 Solutions: SkQ1 in 20% (wt %) glycerol, preparedwith phosphate buffer SkQ1 in 50% (wt %) 1,2-propylene glycol withpyruvic acid SkQ1 in 50% (wt %) 1,2-propylene glycol with lactic acidSolid compositions: SkQ1 with PEG-4000 SkQ1 with dextran SkQ1 withp-aminobenzoic acid (p-ABA) SkQ1 with dextran and p-ABA SkQ1 withmyoinosite SkQ1 with pyruvic acid and Pearlitol 200 SkQ1 with pyruvicacid and microcrystalline cellulose SkQ1 with pyruvic acid and F-Melt CSkQ1 with pyruvic acid and Syloid FP SkQ1 with citric (or tartaric acid,or lactic acid, or glycine) and Pearlitol 200 SkQ1 with citric acid (ortartaric acid, or lactic acid, or glycine) and crocrystalline celluloseSkQ1 with citric acid (or tartaric acid, or lactic acid, or glycine) andF-Melt C SkQ1 with citric acid (or tartaric acid, or lactic acid, orglycine) and Syloid FP SkQ1H₂ (reduced form) Solutions: SkQ1H₂ (0.11M)with ascorbic acid (10 eq) in 55% EtOH SkQ1H₂ (7.4 mM) with ascorbicacid (5 eq) and sorbite (20 wt parts) in 30% 1,2-propylene glycol Solidcompositions: SkQ1H2 (1 eq) with ascorbic acid (>2 molar eq) withPEG-4000 SkQ1H2 (1 eq) with ascorbic acid (>2 molar eq) with dextranSkQ1H2 (1 eq) with ascorbic acid (>10 molar eq) with PEG-4000 SkQ1H2 (1eq) with ascorbic acid (>10 molar eq) with dextran SkQ1H₂ (1 eq) withsorbite (30 wt parts) SkQ1H₂ (1 eq) with ascorbic acid (0-5 eq) andsorbite (30 wt parts) SkQ1H₂ (1 eq) with ascorbic acid (0-5 eq) andglucose (10 wt parts) SkQ1H₂ (1 eq) with ascorbic acid (0-5 eq) andlactose monohydrate (10 wt parts) SkQ1H₂ (1 eq) with ascorbic acid (0-5eq) and Pearlitol 200 (30 wt parts) SkQ1H₂ (1 eq) with ascorbic acid(0-5 eq) and microcrystalline cellulose (30 wt parts) SkQ1H₂ (1 eq) withascorbic acid (0-5 eq) and F-Melt C (30 wt parts) SkQ1H₂ (1 eq) withascorbic acid (0-5 eq) and Syloid FP (30 wt parts)

SkQ1H₂ in the from of light powder was prepared to almost a 100% yieldby the reduction of SkQ1 with ascorbic acid or any other suitablereducing agent in alcohol/water mixture followed by isolation by eitherextraction with chloroform or any other suitable solvent, or byprecipitation from water followed by centrifugal separation, or bycolumn (silica gel) chromatography or by method HPLC RP. The isolatedmaterial was characterized by 1H NMR, LC/MC and elemental analysis data.

The sample was proved to have excellent stability for 1 month at RT orseveral months at 4° C. in darkness under inert atmosphere without anyhumidity access (Table 17). The sample also can be stabilized by beingdissolved in any deoxygenated anhydrous and aprotic solvents. Thereduced form of SkQ1H₂ quickly oxides to the original form of SkQ1 whenexposed to air or wet atmosphere or dissolved in water or any protonicsolvent (Table 18).

The stability of SkQ1H₂ in solid compositions is strongly dependent ondryness of the composition as well as dryness of excipients and othercomponents. Humidity of ambient atmosphere and presence of air also playa crucial role in oxidation of SkQ1H₂ into SkQ1 followed by degradationof the latter.

II. Treatments

In vivo and in vitro experiments demonstrate the ability of MTAsincluding, but not limited to, SkQ1 and SkQR1, to prevent and treatdiabetes and disorders related to diabetes (Example 2). Such in vivo andin vitro experiments also demonstrate that liquid solutions of MTAs,including but not limited to SkQ1 and SkQR1, can be used for preventionand treatment of inflammatory diseases and related conditions such asseptic shock and/or systemic. For example, these MTA-based liquidformulations with acceptable stability combined with results showingefficacy in models of diabetes, inflammation, septic shock, and relateddisorders (Examples 2-7).

SkQ1 treatment also prevented disassembling of intracellular contactsand cytoskeleton reorganization caused by TNFa (data obtained bymicroscopy studies of VE-cadherin, beta-cathenin and F-actin). Thus,SkQ1 was shown to be effective in protecting endothelial cells againstthe cytokine-caused dysfunction of endothelial barrier, and thus can beused for prevention and treatment of many pathological conditionsincluding diabetes, atherosclerosis, aging, and chronicle inflammatorydiseases.

Additionally, SkQ1 decreases the phosphorylation and degradation of IkBacaused by TNFα. NFκB is known to be permanently active in manyinflammatory diseases, such as inflammatory bowel disease, arthritis,sepsis, gastritis, asthma and atherosclerosis (Monaco et al. (2004)PNAS., 101:5634-9). SkQ1 was shown to prevent activation of NFκB, a keyinhibitor of NFκB activity associated with elevated mortality,especially from cardiovascular diseases (Venuraju et al. (2010) J. Am.Coll. Cardiol., 55:2049-61). In addition, SkQ1 was shown to preventtranslocation of transcription factor p65 (RelA) from the cytoplasm tothe nucleus, thereby potentially decreasing pathological consequences.

Reference will now be made to specific examples illustrating theinvention. It is to be understood that the examples are provided toillustrate certain embodiments and that no limitation to the scope ofthe invention is intended thereby.

EXAMPLES Example 1 Stable Formulations of Reduced Form of SkQ1 (SkQ1H₂)

SkQ1H₂, a reduced quinole form of SkQ, was prepared as follows: 10 mlSkQ1H₂ solution (with concentration 1 mg/ml) in ethanol was thoroughlymixed with 200 mg ascorbic acid and then vacuum dryed. The resultingpowder contained 95% ascorbic acid and 5% SkQ1H₂, and demonstratedacceptable stability at several storage temperatures. For example, inthe accelerated decay experiment, SkQ1 purity was reduced from initial98.7% to 95.1% after storage for 12 d at 60° C. From these results itcan be calculated that storage for 1 year at 4° C. will result inapproximately 3.5% loss from the initial concentration of the activecompound SkQ1 which has acceptable stability.

Alternatively, a dry mixture of SkQ1H₂ and ascorbic acid is prepared bydissolving 10 mg SkQ1H₂ in 10 ml ascorbic acid solution (20 mg/ml) anddried under vacuum.

Yet another way to prepare an SkQ1-ascorbic acid mixture is to mix 5 mlSkQ1H₂ solution in ethanol (2 mg/ml), with 5 ml ascorbic acid solutionin water (40 mg/ml), and vacuum dry. The reduced form of SkQH₂ isstabilized in ascorbic acid solution, eliminating the drying stage, andthus the corresponding liquid formulation.

Example 2 Effect of Liquid MTA Formulations on Diabetes A. AlloxanAnimal Studies

Alloxan is a well-known diabetogenic agent widely used to induce type 2diabetes in animals (Viana et al. (2004) BMC Pharmacol., 8:4-9).

Induction of the alloxan diabetes was performed as follows: Two groupsof laboratory rats (20 animals in each group) with free food and wateraccess fed a 250 nM solution of SkQ1 for 10 d. The daily rat consumptionwas 60 ml water solution (containing 15 nmoles SkQ1). The average weightof rats was 300 g. Thus, rats consumed approximately 50 nmol/kg bodyweight per day. Two other groups of animals did not receive SkQ1. After10 d, rats were subcutaneously (in the area of the thigh) injected withalloxan dissolved in isotonic salt solution of 0.9% w/v of NaCl (100mg/kg body weight; groups “Alloxan+SkQ1” and “Alloxan.” Control animalswere injected with salt solution without alloxan (groups “Control+SkQ1”and “Control”). After injection, the rats continued to drink watercontaining SkQ1 (250 nM) during 14 d (group “Alloxan+SkQ1”) or were keptwithout SkQ1 (group “Alloxan”).

Data on glucose blood level was measured by the glucose oxidase method(Saifer et al. (1958) J. Lab. Clin. Med., 51:445-460) after 2 weeks ofalloxan injection. The results are presented in FIG. 1. All data arepresented as the mean+/−SE.

Animals consuming SkQ1 after alloxan injection had about 2-fold lowerblood glucose compared to mice without SkQ1 treatment.

These results demonstrate that stabilized MTAs, e.g. SkQ1, are usefulfor the prevention and treatment of diabetes mellitus and itscomplications.

In another experiment, 200 g to 250 g Wistar male rats (age 7 to 8weeks) were divided into 3 groups, 12 to 15 animals each and wereinjected with alloxan 125 mg/kg intraperitoneally (i.p.) after overnightfasting. Control animals were injected with saline (0.9% NaCl). Thestabilized formulation (1% ethanol, 5 ml/kg) and SkQ1H₂ (5 eq ascorbicacid, 30 wt parts sorbite) in a dosage of 1250 nmol/kg was administeredintragastrically (i.g.) by gavage once daily for 2 weeks before and 1week after alloxan administration. Blood samples from tail vein werecollected after overnight fasting and glucose levels were measuredbefore alloxan administration and 1 d, 2 d, 3 d, and 7 d later by theconventional glucose-oxidase method. Seven days after alloxanadministration rats were subjected to a glucose tolerance test. Ratswere given glucose 3 g/kg i.g. Blood glucose levels were measured beforeglucose injection and 15 min, 30 min, 60 min, and 90 min later.

The following results were obtained (Table 3):

TABLE 3 Saline + Alloxan + SkQ1H₂ vehicle Alloxan + vehicle formulationMaximum glucose 6.7 17.6 13.9 conc. in blood, mM Integrated glucose 5001194 947 con. in blood (area under curve, a.u = mM × min)

B. Diabetic Mouse Studies

Mice carrying mutation in leptin receptor gene (C57BLKS-Leprdb/J mice,or db/db mice) are known to be affected by glucose metabolic disorders.These mice are used as type II diabetes model with many of thecharacteristics of human disease including hyperphagia, hyperglycemia,insulin resistance, progressive obesity (Hummel et al. (1966) Science,153:1127-1128).

SkQ1 in 20% glycerol, as described below in Example 8 (250 nmol/kg perday) was orally administered to 10 to 12 week old homozygous db/db mice(n=8), while vehicle db/db (n=8) and non-diabetic control heterozygousdb/++ (n=5) mice for 12 weeks. The hepatic TBA-reactive substancecontent (MDA) was determined by assay according to the method of Miharaet al. ((1978) Anal. Biochem., 86:271-278).

As shown in FIG. 2, elevated glucose levels induce oxidative stressreflected by the increased MDA levels in the liver of db/db mice. Theincrease of MDA level reflects stimulation of lipid peroxidation whichin turn is considered responsible for the impairment of endothelialcells, capillary permeability, and fibroblast and collagen metabolism,major factors of pathologies associated with diabetes. The stabilizedsolution of SkQ1 significantly lowered MDA levels in the liver ofdiabetic db/db mice, thus indicating decreased rate of lipidperoxidation and decreased damage of the liver.

Example 3 Effect of Stabilized MTA on Wound Healing

Wound healing was studied in two series using 6 months oldC57BLKS-Leprdb/J mice (db/db) homozygous and heterozygousC57BLKS-Leprdb/J mice (db/+) mice. These mice are used as type IIdiabetes model with impaired wound healing (Michaels, et al. (2007)Wound Repair and Regeneration, 15:665-670).

The mice were daily administered 250 nmol/kg body weight per day withthe pharmaceutical form of SkQ1 in 20% glycerol as described in Example8) during period of time from 10 weeks to 12 weeks. Control groups ofdb/db and db/+ mice were not treated with SkQ1. Full-thickness dermalwounds were made under anesthesia of ketamine (80 mg/kg). Animals werekept in plastic cages under standard temperature, light, and feedingregimes. 7 days after wounding, animals were sacrificed by decapitation.The wounds were excised, fixed in 10% formalin in standard PBS buffer,histologically processed, and embedded in paraffin. Histologicalsections of central part of the wounds were cut and stained withhematoxylin and eosin. The sections were immunohistochemically stainedfor markers of endothelial cells (CD31), macrophages (f4/80), andmyofibroblasts (smooth muscle α-actin). ImageJ software (NationalInstitutes of Health (NIH) http:/rsb.info.nih.gov/ij/) was used tocalculate total amount of cells, number of neutrophils, macrophages andvessel density (vessel area/granulation tissue area*100) on themicrophotographs of wound sections. For each animal 100 mm² of sectionarea was analyzed. Wound epithelization rate was assessed in % as ratioof epithelized wound area to total wound area on tissue section*100. Forstatistical analysis nonparametric Mann-Whitney U-test was used. Dataare shown as means±S.E.M.

As shown in FIGS. 3 a, 3 b and 3 c, the stabilized pharmaceutical formof SkQ1 is able to accelerate wound healing by decreasing neutrophilinfiltration, increasing vascularization, and increasing the rate ofepithelization in diabetic mice.

Example 4 Effect of Stabilized MTA on Inflammation and Septic Shock

Septic shock is known to activate numerous inflammatory pathways in anorganism leading to death. The lipopolysaccharide (LPS)-induced septicshock mouse is widely accepted model in pharmacological and biologicalresearch (Villa et al. (2004) Meth. Molec. Med., 98:199-206).

Induction of the septic shock was performed as follows: 43 male BALB/cmice with free food and water access were divided onto 4 experimentalgroups. Group “K” got water without drugs. Groups “SkQ 50,” “SkQ 250,”and “SkQ 1250” were daily parenterally treated with pharmaceutical formof SkQ1 in water comprising 50 nmol/kg, 250 nmol/kg, and 1250 nmol/kgaccordingly. After 3 weeks of SkQ1 treatment animals wereintraperitoneally injected with 250 mg/kg LPS and 700 mg/kgD-galactosamine (D-GalN) inducing septic shock leading to death of 50%of untreated control animals (LD50 dose). Death of animals wereregistered after 4 d of septic shock induction.

The results of the experiment are shown on FIG. 4. The survival of micefollowing LPS/D-GalN treatment was significantly improved by SkQ1. Thestatistically significant effect was shown for a dose of 50 nmol/kg(p=0.03).

These results clearly indicate that SkQ1 acts as an anti-inflammatoryagent having a therapeutic application for septic shock treatment.

In other studies, BALB/c mice with free food and water access aredivided onto 4 experimental groups. Group “K” receive 20% glycerolwithout drugs. Groups “SkQ 50,” “SkQ 250,” and “SkQ 1250” are dailyparenterally treated with pharmaceutical form of SkQ1 in 20% glycerol(Example 8) comprising 50 nmol/kg, 250 nmol/kg, and 1250 nmol/kgaccordingly. After 3 weeks of SkQ1 treatment animals areintraperitoneally injected with 250 mg/kg LPS and 700 mg/kgD-galactosamine (D-GalN) inducing septic shock leading to death of 50%of untreated control animals (LD50 dose). Death of the animals isregistered after 4 d of septic shock induction.

Example 5 Effect of Stabilized MTA on Arthritis

The collagen-induced arthritis (CIA) rat model was used to examine thesusceptibility of rheumatoid arthritis (RA) to treatment with potentialanti-arthritic agents (Griffiths et al. (2001) Immunol. Rev.,184:172-83).

Thirty Wistar rats with free food and water access were injected withcomplete Freund adjuvant and 250 mg type II collagen to induce CIA.Starting from 14 d and from 24 d after injection, two groups of 10animals in each were daily fed with pharmaceutical form of SkQ1 in watercomprising 250 nmol/kg body weight per day (groups “SkQ1 from day 14”and “SkQ1 from day 24”; Group “Control” received water without drugs).

As shown in FIG. 5, SkQ1 reduced the number of animals with apparentinflammation, i.e. animals with increased paw volumes measured by watermanometry compared to control group. Hence, SkQ1 possessesanti-inflammatory and anti-arthritic effects.

In other studies, Wistar rats with free food and water access areinjected with complete Freund adjuvant and 250 mg type II collagen toinduce CIA. Starting from 14 d and from 24 d after injection, two groupsof animals in each are daily fed with pharmaceutical form of SkQ1 in 20%glycerol (Example 8) comprising 250 nmol/kg body weight per day (groups“SkQ1 from day 14” and “SkQ1 from day 24”; Group “Control” receivedwater without drugs).

Example 6 Effect of Stabilized MTA on Inflammation Associated withCoronary Heart Disease

Intense cytokine production induced by inflammation may lead to death ofendothelial cells which, along with increased oxidative stress andvascular inflammation, leads to endothelial dysfunction and increasesthe risk for coronary artery disease.

Human endothelial cell line EA.hy926 (ATCC Collection; catalog numberCRL-2922) was used as a model of vascular endothelium. This cell line issimilar to primary HUVEC cell line (Edgell et al. (1983) PNAS,80(12):3734-7; Edgell et al. (1990) In Vitro Cell Dev Biol.,26(12):1167-72) and widely used as a relevant model for inflammationstudies (Riesbeck et al. (1998) Clin. Vaccine Immunol., 5:5675-682).

Accordingly, human endothelial cells EA.hy926 were pre-incubated with0.2 nM SkQR1 or 2 nM SkQ1 solution in Dulbecco's Modified Eagle's Medium(DMEM) supplemented with 10% of fetal serum (Example 1) for 4 d. Afterthat the cells were incubated overnight with fresh DMEM medium with 0.2%of fetal serum. The cells were incubated 2 d with TNF-α (0.25 ng/ml to50 ng/ml) and cell death was monitored using standard MTT test (Berridgeet al. (1996) Biochemica, 4:14-9). The data from this assay is shown asmeans±S.E. at least for 3 separate experiments.

As shown in FIG. 6, both SkQ1 and SkQR1 greatly reduced cell deathcompared to control without MTA. Thus, SkQ1 and SkQR1 were shown to beeffective substance protecting endothelial cells against cytokine'sinflammatory action and can be used for prevention and treatment ofcoronary heart disease including atherothrombosis.

Example 7 Effect of Stabilized MTA on Vascular Dysfunction A. In VitroStudies

Inflammatory cytokines induce expression of ICAM-1 (Inter-CellularAdhesion Molecule 1). ICAM-1 is a key molecule functioning inintercellular adhesion process and transmigration of leukocytes acrossvascular endothelia during inflammatory response. Expression of ICAM-1,as well as inflammatory cytokines including IL-6 and IL-8, is elevatedunder many pathological conditions including diabetes, atherosclerosis,aging, and chronicle inflammatory diseases.

The effects of SkQ1 on ICAM-1 mRNA expression and cytokines (IL-6, IL-8)protein secretion induced by TNF-α in EAhy926 human endothelial cells(ATCC collection; catalog number CRL-2922) were examined. TNF-α is acentral proinflammatory cytokine stimulating expression of cell adhesionmolecules and many inflammatory cytokines. Anti-inflammatory propertiesof many drugs often rely on their ability to inhibit expression ofpro-inflammatory cytokines induced by TNF-α using EAhy926 endothelialcells (Edgell et al. (1983) Proc. Natl. Acad. Sci. USA, 80:3734-7;Lombardi et al. (2009) Eur. J. Cell. Biol., 88:731-42; Manea et al.(2010) Cell Tissue Res., 340:71-9).

300,000 cells were plated on 60 mm² culture dishes and after attachmentwere treated with an SkQ1 solution (0.2 nM in DMEM medium with 10% fetalserum) for 4 d, and then stimulated with TNF-α (0.05 ng/ml for 4 h forICAM-1 or 5 ng/ml for 15 h for cytokines, respectively). ICAM-1 mRNAexpression was determined by real-time PCR (Okada et al. (2005) Invest.Ophtalmol. Vis. Sci., 46:4512-8). Secretion of IL-6 and IL-8 wasevaluated by ELISA (Toma et al. (2009) Biochem. Biophys. Res. Commun.,390:877-82; Volanti et al. (2002) Photochem. Photobiol., 75:36-45.) Thedata is shown as means±S.E. at least for 3 separate experiments.

The results shown in FIG. 7 a confirm SkQ1 to be and effective vascularanti-inflammatory substance that prevents excessive expression ofinflammatory cytokines and ICAM-1. Thus, MTAs are useful for preventionand treatment of vascular pathologies including atherosclerosis.

B. In Vivo Studies

As described above in Example 7A, above, the expression of ICAM-1 iselevated under many pathological vascular conditions. SkQ1 efficacy inreducing ICAM-1 expression in vivo was tested on mice. 30 hybrid maleC57Black/CBA mice were divided into 3 experimental groups (10 animals ineach group) at the beginning of the experiment. The group “Young mice”included mice at the age of 6 months. Groups “Old mice” and “Old mice,SkQ1” included mice at the age of 24 months. The group “Old mice, SkQ1”had free access to drinking water with 100 nM water-dissolved SkQ1 per 1kg of body weight for 7 months. After this period, the animals weredecapitated. Aortas were excised, and total RNA was isolated usingDNeasy Blood and Tissue kit (QIAGEN), reverse-transcribed into cDNA, andused for quantitative real-time PCR analysis of ICAM-1 mRNA level. Forthe normalization procedure the average geometry of expression levels ofhousekeeping genes GAPDH and RPL32 was used Data are shown asmeans±S.E.M.

As shown on FIG. 7 b, SkQ1 significantly lowered ICAM-1 mRNA levels intreated old mice compared to the control group and approaches the levelof ICAM-1 in young mice.

The results demonstrate that SkQ1 prevents the age-related increase ofICAM-1 expression in the vascular endothelium. Thus, SkQ1 can be usedfor prevention of age-related vascular pathologies includingatherosclerosis.

In other studies, hybrid male C57Black/CBA mice are divided into 3experimental groups, “young,” “old,” and “old mice, SkQ1,” as describedabove. The third group receives SkQ1 in 20% glycerol comprising 250nmol/kg body weight per day dose up to 7 months. The “old” group is thecontrol and receives glycerol without drugs. After this period, theanimals are decapitated. Aortas are excised, and total RNA is isolatedusing DNeasy Blood and Tissue kit (QIAGEN), reverse-transcribed intocDNA, and used for quantitative real-time PCR analysis of ICAM-1 mRNAlevel. For the normalization procedure the average geometry ofexpression levels of housekeeping genes GAPDH and RPL32 are used. Dataare calculated as means±S.E.M.

Example 8 Preparation and Stability of Oxidized SkQ1 Formulations 1.SkQ1 in 20% (wt %) Glycerol and Phosphate Buffer

Glycerol (20 g) was diluted with phosphate buffer (80 g, 0.01 M KH₂PO₄,pH 4.77). A sample of SkQ1 (20 mg) was placed in a dark glass vial anddissolved in propylene glycol (0.2 mL) and diluted with an aliquot (19.8ml) of the above solvent to 1 mM.

The stability of SkQ1 in the prepared solution was investigated bystorage at RT and at 60° C. (Table 4).

TABLE 4 SkQ1, %/degradation SkQ1, %/degradation Time, days products, %(stored at RT) products, % (stored at 60° C.) 0 99.34/0   99.34/0   1199.71/0   — 13 99.76/0   — 14 99.68/0   — 17 99.62/0   — 19 99.63/0.0795.30/4.7 21 99.52/0.20 — 24 99.57/0.08 — 61 99.49/0.51 —2. SkQ1 in 50% (wt %) 1,2-Propylene Glycol with Pyruvic Acid (10Equivalents (eq) Relative to SkQ1)

SkQ1 (50 mg) and pyruvic acid (71 mg, 10 eq) were placed in a dark glassvial and dissolved in 50% propylene glycol-water mixture (100 ml) toyield a 0.081 mM SkQ1 solution.

The stability of SkQ1 in the prepared solution was investigated bystorage at 60° C. (Table 5).

3. SkQ1 in 50% (wt %) 1,2-Propylene Glycol with Lactic Acid (10 eqRelative to SkQ1)

SkQ1 (50 mg) and L(+)-lactic acid (73 mg, 10 eq) were placed in a darkglass vial and dissolved in 50% propylene glycol-water mixture (100 ml)to yield a 0.081 mM SkQ1 solution.

The stability of SkQ1 in the prepared solution was investigated bystorage at 60° C. (Table 5).

TABLE 5 Time, days SkQ1, % SkQ1, % 0 >99.9 >99.9 72 93.2 96.64. SkQ1 with PEG-4000

A solution of 8 mg SkQ1 in 0.5 ml EtOH was mixed with 200 mg PEG-4000,and the solvent was evaporated to dryness.

The stability of SkQ1 in the prepared composition was investigated bystorage at 4° C. in darkness (Table 6).

TABLE 6 Time, days SkQ1, % Degradation products, % 18 >99.9 <0.01 1999.83 0.17 20 99.80 0.205. SkQ1 with Dextran

A solution of 10 mg SkQ1 in 0.75 ml EtOH was added to a solution of 100mg dextran in 1 ml water. The mixture was vigorously stirred and thesolvent was evaporated to dryness.

The stability of SkQ1 in the prepared composition was investigated bystorage at 60° C. in darkness (Table 7).

TABLE 7 Time, days SkQ1, % Degradation products, % 0 96.71 3.29 6 20.6679.34 15 24.14 75.86 25 18.93 81.076. SkQ1 with p-Aminobenzoic Acid (p-ABA)

A solution of 8 mg SkQ1 in 0.5 ml EtOH was added to a solution of 200 mgp-aminobenzoic acid (p-ABA) in 1.5 ml EtOH. The solvent was evaporatedto dryness.

The stability of SkQ1 in the prepared composition was investigated bystorage at RT in darkness (Table 8).

TABLE 8 Time, days SkQ1, % Degradation products, % 0 100 0 30 58.4241.587. SkQ1 with Dextran and p-ABA

A solution of 10 mg SkQ1 in 0.75 ml EtOH was added to a solution ofp-ABA (2 mg in 0.5 ml EtOH) and dextran (100 mg in 1 ml water). Themixture was vigorously stirred and the solvent was evaporated todryness.

The stability of SkQ1 in the prepared composition was investigated bystorage at 60° C. in darkness (Table 9).

TABLE 9 Time, days SkQ1, % Degradation products, % 0 97.13 2.87 6 39.2260.78 15 7.07 92.938. SkQ1 (1 eq) with Myoinosite (30 wt Parts Relative to SkQ1)

45 mg myoinosite was added to a solution of 5 mg SkQ1 in 5 ml EtOH. Themixture was vigorously stirred and the solvent was evaporated todryness.

The stability of SkQ1 in the prepared composition was investigated bystorage at RT in darkness (Table 10).

TABLE 10 Time, days SkQ1, % Degradation products, % 0 95.88 4.12 5 96.863.14 6 95.99 4.01 15 92.26 7.749. SkQ1 (1 eq) with Pyruvic Acid (10 eq) and Pearlitol 200 (30 wt PartsRelative to SkQ1)

375 mg Pearlitol 200 was added to a solution of 12.5 mg SkQ1 and 17.8 mg(10 eq) pyruvic acid in 0.75 ml EtOH. The mixture was vigorously stirredand the solvent was evaporated to dryness.

The stability of SkQ1 in the prepared composition was investigated bystorage at 60° C. in darkness (Table 11).

10. SkQ1 (1 eq) with Pyruvic Acid (10 eq) and Microcrystalline Cellulose(30 wt Parts Relative to SkQ1

375 mg microcrystalline cellulose was added to a solution of 12.5 mgSkQ1 and 17.8 mg (10 eq) pyruvic acid in 0.75 ml EtOH. The mixture wasvigorously stirred and the solvent was evaporated to dryness.

The stability of SkQ1 in the prepared composition was investigated bystorage at 60° C. in darkness (Table 11).

11. SkQ1 (1 eq) with Pyruvic Acid (10 eq) and F-Melt C (wt PartsRelative to SkQ1)

375 mg F-Melt C was added to a solution of 12.5 mg SkQ1 and 17.8 mg (10eq) pyruvic acid in 0.75 ml EtOH. The mixture was vigorously stirred andthe solvent was evaporated to dryness.

The stability of SkQ1 in the prepared composition was investigated bystorage at 60° C. in darkness (Table 11).

12. SkQ1 (1 eq) with Pyruvic Acid (0 eq) and Syloid FP (30 wt PartsRelative to SkQ1)

375 mg Syloid FP was added to a solution of 12.5 mg SkQ1 and 17.8 mg (10eq) pyruvic acid in 0.75 ml EtOH. The mixture was vigorously stirred andthe solvent was evaporated to dryness.

The stability of SkQ1 in the prepared composition was investigated bystorage at 60° C. in darkness (Table 11).

TABLE 11 Time, SkQ1, %/SkQ1H₂, %, degradation products, % days (Sample9) (Sample 10) (Sample 11) (Sample 12)0 >99.9/<0.05/ >99.9/<0.05/ >99.9/<0.05/<0.05 >99.9/ <0.05 <0.05 <0.05/<0.05 14 60.3/11.3/ 50.2/25.8/24.0 38.2/47.7/14.1 57.9/1.4/ 28.4 40.7

The following SkQ1 preparations can also be formulated as describedsupra in Example 8:

-   -   SkQ1 (1 eq) with citric (or tartaric acid, or lactic acid, or        glycine, 10 eq) and Pearlitol 200 (30 wt parts in relation to        SkQ1H₂)    -   SkQ1 (1 eq) with citric acid (or tartaric acid, or lactic acid,        or glycine, 10 eq) and microcrystalline cellulose (30 wt parts        in relation to SkQ1H₂)    -   SkQ1 (1 eq) with citric acid (or tartaric acid, or lactic acid,        or glycine, 10 eq) and F-Melt C (30 wt parts in relation to        SkQ1H₂)    -   SkQ1 (1 eq) with citric acid (or tartaric acid, or lactic acid,        or glycine, 10 eq) and Syloid FP (30 wt parts in relation to        SkQ1H₂)

Example 9 Preparation and Stability of Reduced SkQH₂ Formulations 13.SkQ1H2 (1 eq) Prepared in Situ by Reduction of SkQ1 and Ascorbic Acid (2Molar eq) and PEG-4000 (10 wt Parts Relative to SkQ1H₂)

A solution of 10 mg SkQ1 in 0.6 ml EtOH was added to solution of 5.7 mg(2 eq) ascorbic acid in 0.1 ml water. The mixture was stirred untilreduction to SkQ1H₂ completed (about 1 h). Then 100 mg PEG-4000 wasadded. The mixture was vigorously stirred for 30 min and the solventevaporated to dryness.

The stability of SkQ1H₂ in the prepared composition was investigated bystorage at 4° C. in darkness (Table 12).

14. SkQ1H2 (1 eq Prepared in Situ by Reduction of SkQ1 with AscorbicAcid (2 Molar eq) and Dextran)

A solution of 10 mg SkQ1 in 0.6 ml EtOH was added to solution of 5.7 mg(2 eq) ascorbic acid in 0.1 ml water. The mixture was stirred untilreduction to SkQ1H₂ completed (about 1 h). Then a solution of 100 mgdextran in 1 ml water was added. The mixture was vigorously stirred for30 min and the solvent was evaporated to dryness.

The stability of SkQ1H₂ in the prepared composition was investigated bystorage at 4° C. in darkness (Table 12).

TABLE 12 (Sample 13) (Sample 14) Degra- Degra- dation dation Time, SkQ1,SkQ1H₂, products, SkQ1, SkQ1H₂, products, days % % % % % % 0 14.65 85.35<0.05 3.61 96.39 <0.05 1 7.72 92.28 2.80 97.20 4 59.12 40.88 98.57 1.436 57.53 42.47 99.55 0.45 7 54.16 45.84 99.26 0.74 10 54.22 45.78 98.931.0715. SkQ1H2 (1 eq) Prepared in Situ by Reduction of SkQ1 with AscorbicAcid (10 Molar eq) and Dextran (10 wt Parts Relative to SkQ1H₂)

A solution of 10 mg SkQ1 in 0.6 ml EtOH was added to solution of 28.5 mg(10 eq) ascorbic acid in 0.25 ml water. The mixture was stirred untilreduction to SkQ1H₂ was completed (about 30 min). A solution of 100 mgdextran in 1 ml water was then added. The mixture was vigorously stirredfor 30 min and the solvent evaporated to dryness.

The stability of SkQ1H₂ in the prepared composition was investigated bystorage at 60° C. in darkness (Table 13).

16. SkQ1H2 (1 eq) (Prepared in Situ by Reduction of SkQ1 with AscorbicAcid (>10 Molar eq) with Dextran and p-ABA (10 wt Parts Relative toSkQ1H₂

A solution of 10 mg SkQ1 in 0.6 ml EtOH was added to solution of 28.5 mg(10 eq) ascorbic acid in 0.25 ml water. The mixture was stirred untilreduction to SkQ1H₂ was completed (about 30 min). A solution of 100 mgdextran in 1 ml water and a solution of 2 mg p-ABA in 0.5 ml EtOH werethen added. The mixture was vigorously stirred for 30 min and thesolvent evaporated to dryness.

The stability of SkQ1H₂ in the prepared composition was investigated bystorage at 60° C. in darkness (Table 13).

TABLE 13 (Sample 15) (Sample 16) Degra- Degra- dation dation Time, SkQ1,SkQ1H₂, products, SkQ1, SkQ1H₂, products, days % % % % % % 0 2.35 92.595.06 0.74 98.65 0.61 6 4.26 91.66 4.08 2.72 97.16 0.12 15 5.11 94.270.62 8.49 91.12 0.39 25 5.71 88.69 5.6 11.07 86.62 2.31

17. SkQ1H₂ Powder

A solution of 2 g SkQ1 in 40 ml EtOH was added to a solution of 5.7 gascorbic acid in 60 ml water. The mixture was stirred until reduction toSkQ1H₂ was completed (about 30 min). Completion of reduction can bedetected as the solution becomes colorless. The solvent was thenevaporated off and the residue was partitioned between water (50 ml) andCHCl₃ (150 ml). The organic layer was washed with water (2×25 ml), driedwith anhydrous sodium sulfate, filtered, and evaporated.

The yield of SkQ1H₂ was 2 g (approx 100% yield) in the form of lightpowder. The stability results are shown below (Table 14 and Table 15).

TABLE 14 Storage at RT Storage at 60° C. Degra- Degra- dation dationTime, SkQ1H₂, SkQ1, products, SkQ1H₂, SkQ1, products, days % % % % % % 098.99 1.01 <0.1 99.2 0.75 0.05 3 99.34 0.66 <0.1 — — — 5 99.37 0.63 <0.199.45 0.55 0 7 99.14 0.71 <0.1 100 0 0 11 99.12 0.83 <0.1 99.76 0.190.05 17 99.49 0.28 <0.3 98.61 1.24 0.15 28 99.45 0.50 <0.1 88.7 11.040.26

TABLE 15 55% EtOH in water CH₂Cl₂ Degra- Degra- dation dation Time,SkQ1H₂, SkQ1, prod- SkQ1H₂, SkQ1, prod- h % % ucts, % % % ucts, % 097.79 2.21 0 97.79 2.21 0 0.5 90.79 9.21 0 95.99 4.01 0 1.48 85.24 14.760 94.32 5.68 0 2.8 67.43 32.57 0 93.58 6.42 0 3.44 52.17 47.83 0 94.435.57 0 4.37 43.43 56.57 0 92.82 7.18 0 23.23 16.55 82.39 1.06 89.61 9.300.97 143.45 9.63 77.23 13.14 82.11 16.53 1.36 (~6 days)18. SkQ1H₂ (1 eq) with Sorbite (30 wt Parts Relative to SkQ1H₂)

A solution of 20 mg SkQ1H₂ in 1.3 ml EtOH was added to a solution of 600mg sorbite in 1.3 ml water. The solvent was evaporated to dryness. Theresidue was additionally dried with diphosphorous pentoxide (P₂O₅) underreduced pressure.

The stability of SkQ1H₂ in the prepared composition was investigated bystorage at 60° C. in darkness (Table 16).

TABLE 16 Time, days (at 60° C.) SkQ1H₂, % SkQ1, % Degradation products,% 0 99.01 0.61 0.38 4 90.8 8.7 0.5 7 90.2 9.4 0.4 11 88.8 10.7 0.5 1589.1 10.4 0.5 28 42.9 5.3 51.819. SkQ1H₂ (1 eq) with Ascorbic Acid (0-5 eq) and Sorbite (30 wt PartsRelative to SkQ1H₂)

Method 1:

A solution of 20 mg SkQ1H₂ in 1.3 ml EtOH was added to a solution of28.4 mg (5 eq) ascorbic acid and 600 mg sorbite in 1.3 ml water. Thesolvent was evaporated to dryness. The residue was additionally driedwith P₂O₅ under reduced pressure.

Method 2:

20 mg SkQ1H₂ and 28.4 mg (5 eq) ascorbic acid were added to sorbite (600mg) melted in a glass vial (bath temperature 110° C.) slowly undervigorous stirring and stirring continued for 1 hr. The mixture wascooled to RT and vigorously triturated to provide a microcrystallinepowder.

The stability of SkQ1H₂ in the compositions prepared by both methods wasinvestigated by storage at 60° C. and 4° C. in darkness (Table 17).

TABLE 17 Degradation products, (total, %/number of impurities withcontent >0.5%) SkQH₂, % SkQ1, % 20 days at 60° C. 1 year at 4° C. 97.7531.209 1/0 0.3/0

The following SkQ1H₂ preparations in ascorbic acid are also prepared asin Example 19 supra:

-   -   SkQ1H₂ (1 eq) with ascorbic acid (0-5 eq) with magnesium        stearate (10 wt % in relation to SkQ1H₂) and glucose (10 wt        parts in relation to SkQ1H₂)    -   SkQ1H₂ (1 eq) with ascorbic acid (0-5 eq) with magnesium        stearate (10 wt % in relation to SkQ1H₂) and lactose monohydrate        (10 wt parts in relation to SkQ1H₂)    -   SkQ1H₂ (1 eq) with ascorbic acid (0-5 eq) and Pearlitol 200 (30        wt parts in relation to SkQ1H₂)    -   SkQ1H₂ (1 eq) with ascorbic acid (0-5 eq) and microcrystalline        cellulose (30 wt parts in relation to SkQ1H₂)    -   SkQ1H₂ (1 eq) with ascorbic acid (0-5 eq) and F-Melt C (30 wt        parts in relation to SkQ1H₂)    -   SkQ1H₂ (1 eq) with ascorbic acid (0-5 eq) and Syloid FP (30 wt        parts in relation to SkQ1H₂)        20-22 and 26-30. SkQ1H₂ with Ascorbic Acid (0-5 eq) and Glucose

Method 3:

A solution of 20 mg SkQ1H₂ in 1.3 ml EtOH was added to 2 mg magnesiumstearate and solution of ascorbic acid (quantities as listed in theTable 18) and 600 mg glycose in 1.3 ml water (1.3 mL). The solvent wasevaporated to dryness. The residue was additionally dried with P₂O₅under reduced pressure.

Method 4:

20 mg SkQ1H₂, 2 mg magnesium stearate, ascorbic acid (quantities aslisted in Table 18) and 600 mg anhydrous glycose were mixed andvigorously triturated.

The stability of SkQ1H₂ in compositions prepared by Methods 3 and 4 wasinvestigated by storage at 60° C. in darkness (Table 18).

23.-25. SkQ1H₂ with Ascorbic Acid (0-5 eq) and Lactose Monohydrate

The compositions were prepared as described above in Method 3 or 4 usinglactose monohydrate instead of glycose.

The stability of SkQ1H₂ in compositions prepared by both methods wasinvestigated by storage at 60° C. in darkness (Table 18).

TABLE 18 Formulation Degradation products, total, (stabilizers andexcipients, amounts %/number of impurities are given in relation toSkQ1H₂ with content >0.5% Sample Asc. acid, L (+)- Mg Method of 20 d at1 year at No eq Glycose Lactose × H₂O Stearate preparation 60° C. 4° C.22 1 ~10 wt parts — 10 wt % 4 >30/7 ~6/2 23 3 ~10 wt parts — 10 wt %4 >12/9  <3/1 24 0.3 ~10 wt parts — 10 wt % 4  >9/7  <3/1 25 1 — ~10 wtparts 10 wt % 4 >12/7 4.6/1 26 3 — ~10 wt parts 10 wt % 4  >9/6  <3/2 270.3 — ~10 wt parts 10 wt % 4 >10/5 3.9/2 28 1 ~10 wt parts — 10 wt % 3 ~6/3 2.8/0 29 2 ~10 wt parts — 10 wt % 3  4.4/1 2.6/0 30 3 ~10 wt parts— 10 wt % 3  4.2/0  2/0 31 5 ~10 wt parts — 10 wt % 3  3.6/0 1.6/0 320.3 ~10 wt parts — 10 wt % 3 3.5/3 — (7 d at 60° C.)31. SkQ1H₂ with Ascorbic Acid in 55% EtOH

A solution of pure SkQ1H₂ (1 g in 5 ml EtOH) was added to solution ofascorbic acid (2.85 g (10 eq) in 10 ml water).

The stability of SkQ1H₂ in the prepared solution was investigated bystorage at RT in darkness (Table 19).

TABLE 19 Time, h SkQ1H₂, % SkQ1, % Degradation products, % 0   99.730.27 <0.01 1.5 99.07 0.93 <0.01  68 (~3 days) 99.05 0.59 <0.4 118 (~5days) 99.69 0.31 <0.01 165 (~7 days) 99.74 0.26 <0.0132. SkQ1H₂ with Ascorbic Acid and Sorbite in 30% 1,2-Propylene Glycol

A solution of pure SkQ1H₂ (50 mg in 1 ml 1,2-propylene glycol) was addedto solution of ascorbic acid (67.4 mg (5 eq)) and sorbite (1.5 g) in 10ml water.

The stability of SkQ1H₂ in the prepared solution was investigated bystorage at 60° C. in darkness (Table 20).

TABLE 20 Time, days SkQ1, % SkQH₂, % Degradation products, % 0 0.1899.82 0.00 3 1.03 98.67 0.30 14 28.34 69.51 2.15 27 51.9 3.2 44.9

Equivalents

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

1. A pharmaceutical formulation comprising a compound of Formula I inoxidized and/or reduced form in about 10% to about 100% of a liquidsolvent selected from the group consisting of glycol, ethanol, andglycerol, with the proviso that in compound of Formula I, A is notubiquinone (e.g., 2-methyl-4,5-dimethoxy-3,6-dioxo-1,4-cyclohexadienyl)or tocopherol or a mimetic of superoxide dismutase or ebselen; when L isdivalent decyl, divalent pentyl, or divalent propyl radical and when Bis triphenylphosphonium cation.
 2. The pharmaceutical formulation ofclaim 1, wherein the compound is reduced.
 3. The pharmaceuticalformulation of claim 1, wherein the compound is oxidized.
 4. Thepharmaceutical formulation of claim 1, wherein the compound is SkQ1 orSkQ1H₂.
 5. The pharmaceutical formulation of claim 1, wherein thecompound is SkQR1 or SkQR1H₂.
 6. The pharmaceutical formulation of claim1, wherein the compound is SkQ3 or SkQ3H₂.
 7. The pharmaceuticalformulation of claim 1, wherein the compound is SkQRB or SkQRBH₂.
 8. Thepharmaceutical formulation of claim 1, wherein the compound is SkQB1 orSkQB1H₂.
 9. The pharmaceutical formulation of claim 1, wherein thecompound is SkQBP1 or SkQBP1H₂.
 10. The pharmaceutical formulation ofclaim 1, wherein the solvent is glycerol.
 11. The pharmaceuticalformulation of claim 1, wherein the solvent is glycerol.
 12. Thepharmaceutical formulation of claim 1, wherein the solvent is ethanol.13. A pharmaceutical formulation comprising: a compound of Formula I inoxidized or reduced form; 1 molar equivalent to 200 molar equivalents ofan antioxidation agent that reduces the oxidized form of the compound ofFormula 1; and a pharmaceutically acceptable carrier.
 14. Thepharmaceutical formulation of claim 13, wherein the antioxidation agentcomprises ascorbic acid.
 15. The pharmaceutical formulation of claim 14,wherein the pharmaceutically acceptable carrier comprises sorbite,glucose, and/or magnesium stearate.
 16. A method of treating diabetestype I or type II, comprising orally administering to a patient in needthereof a therapeutically effective amount of a stabilized compound ofFormula I in liquid or solid form, with the proviso that in compound ofFormula I, A is not ubiquinone (e.g.,2-methyl-4,5-dimethoxy-3,6-dioxo-1,4-cyclohexadienyl) or tocopherol or amimetic of superoxide dismutase or ebselen; when L is divalent decyl,divalent pentyl, or divalent propyl radical and when B istriphenylphosphonium cation.
 17. The method of claim 16, wherein type IIdiabetes is treated with a formulation comprising SkQ1H₂ ascorbic acid,and sorbite.
 18. A method of treating dermal wounds, comprising orallyadministering to a patient in need thereof a therapeutically effectiveamount of a formulation comprising a compound of Formula I, in liquid orsolid form, with the proviso that in compound of Formula I, A is notubiquinone (e.g., 2-methyl-4,5-dimethoxy-3,6-dioxo-1,4-cyclohexadienyl)or tocopherol or a mimetic of superoxide dismutase or ebselen; when L isdivalent decyl, divalent pentyl, or divalent propyl radical and when Bis triphenylphosphonium cation.
 19. The method of claim 18, wherein theformulation comprises SkQ1 in 20% glycerol.
 20. A method of treating aninflammatory disorder, comprising orally administering to a patient inneed thereof a therapeutically effective amount of formulationcomprising a stabilized compound of Formula I in liquid or solid form,with the proviso that in compound of Formula I, A is not ubiquinone(e.g., 2-methyl-4,5-dimethoxy-3,6-dioxo-1,4-cyclohexadienyl) ortocopherol or a mimetic of superoxide dismutase or ebselen; when L isdivalent decyl, divalent pentyl, or divalent propyl radical and when Bis triphenylphosphonium cation.
 21. The method of claim 20, wherein theinflammatory disorder is arthritis.
 22. The method of claim 21, whereinthe formulation comprises SkQ1 in 20% glycerol.